We are interested in determining how the protein environment affects the properties of a redox active center; we have chosen to study two classes of proteins containing two types of centers, flavoproteins and dinuclear iron proteins, both of which catalyze electron transfer reactions. Our goals are to relate the redox potentials to protein function, and determine how this function is further 'tuned' by substrate/component binding. Ultimately, we seek to identify the specific structural features of the active site which govern enzyme function and regulation. Members of both protein classes share common redox characteristics: 1) they have redox-active centers that participate in catalysis; 2) they are capable of one- or two-electron transfer, utilizing unique radical of mixed valent intermediates; 3) protonation accompanying electron transfer is important in thermodynamic regulation; and 4) their redox properties appear to be regulated by the binding of substrate, product, or regulatory protein components. The structures of members of both groups are either known by X-ray crystallography or are under intense study by other spectroscopic methods. The electrochemical data we are proposing to accumulate is essential to progress in the study of both classes of proteins. The investigation of both groups of proteins will increase our ability to construct structure/function relationships applicable to each set of proteins. For our redox studies, we will focus on two structurally well characterized proteins from each class with structural and mechanistic similarities: short chain acyl-CoA dehydrogenase (SCAD) and medium chain acyl-CoA dehydrogenase (MCAD) for the flavoproteins; ribonucleotide reductase (RNR) and methane monooxygenase hydroxylase (MMO) for the dinuclear iron proteins. Electrochemical measurements, mutated proteins, and substrate/product analogs, together with well characterized model systems, serve as our tools for defining those protein structural features which govern the feasibility and/or mechanism of electron transfer. Redox data has provided the clearest evidence to date that the electron transfer of three important flavoproteins is thermodynamically controlled by substrate/product binding. Our redox measurements have shown that substrate/product binding to both MCAD and SCAD (two key enzymes in beta- oxidation) provides the thermodynamic driving force for the electron transfer reaction. Studies on the dinuclear iron proteins have progressed to the point where redox data is critical in solving the mechanism of electron transport and the features of the active site which influence the mode of reactivity. Redox studies have already played a crucial role in contributing to the structure of the dinuclear iron protein uteroferrin and in modifying a proposed mechanism for inhibitor binding to this protein. There is strong spectroscopic evidence that electron transfer in dinuclear iron proteins, including RNR and MMO will be regulated by substrate/component binding.